ROLE OF THR-252 IN CYTOCHROME P450(CAM) - A STUDY WITH UNNATURAL AMINO-ACID MUTAGENESIS
ROLE OF THR-252 IN CYTOCHROME P450(CAM) - A STUDY WITH UNNATURAL AMINO-ACID MUTAGENESIS
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DOI：
10.1006/bbrc.1995.1310
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发表时间：
1995-03-08
影响因子：
3.1
通讯作者：
ISHIMURA, Y
中科院分区：
文献类型：
--
作者：
KIMATA, Y;SHIMADA, H;ISHIMURA, Y
通过常规定点诱变用非羟基氨基酸残基如Ala和瓦尔取代细胞色素P450 cam活性中心中的Thr-252，将该单加氧酶转化为NADH氧化酶（Imai，M.等人，Proc. Natl. Sci.联合S. A. 86，7823-7827，1989）。在该研究中，通过非天然氨基酸诱变的方法（Noren，C. J.等人，Science 244，182-188，1989）。与其他定点突变体没有羟基的位置，OMe-突变体保留了相当高的单加氧酶活性，产生化学计量的5-外羟基樟脑的消耗的氧。因此，在这个位置上的游离羟基不是如先前所提出的单加氧酶裂解分子O-2的O-O键的必不可少的必要条件。(C)北京：科学出版社. Inc.
Replacement of Thr-252 in the active center of cytochrome P450cam with a non-hydroxy amino acid residue such as Ala and Val by conventional site-directed mutagenesis converted this monooxygenase to an NADH oxidase (Imai, M. et al. Proc. Natl. Sci. U. S. A. 86, 7823-7827, 1989). In this study, a mutant enzyme with a methoxy group in place of the hydroxy group of Thr-252 (OMe-mutant) was synthesized by the method of unnatural amino acid mutagenesis (Noren, C. J. et al., Science 244, 182-188, 1989). Unlike other site-directed mutants without a hydroxy group at the position, the OMe-mutant retained a considerably high monooxygenase activity, yielding a stoichiometric amount of 5-exo-hydroxycamphor to that of the oxygen consumed. Thus a free hydroxy group at this position is not an indispensable requisite for the monooxygenase to cleave the O-O bond of molecular O-2 as previously proposed. (C) 1995 Academic Press. Inc.